MIT OpenCourseWare http://ocw.mit.edu5.36 Biochemistry LaboratorySpring 2009For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms.SESSION 13 and SESSION 14 (lab open 1-5 pm each day) Sign up for UV/Vis time with your TA. The class will be split into a 1-3 or a 3-5 pm slot. During laboratory Sessions 13 and 14 you will • Use a coupled phosphorylation assay to determine the specific activity of the wild type Abl kinase domain 1) in the absence of an inhibitor 2) in the presence of the drug Gleevec (FDA approved in 2001) and 3) in the presence of the drug Dasatinib (FDA approved in 2006) • Use a coupled phosphorylation assay to determine the specific activity of the H396P Abl kinase domain 1) in the absence of an inhibitor 2) in the presence of the drug Gleevec and 3) in the presence of the drug Dasatinib Researchers use a variety of strategies to monitor the activity of kinases. You will recall that kinases are enzymes that catalyze the phosphorylation of substrate peptides or proteins by transferring a phosphate group from a molecule of ATP to the substrate. Phosphorylation is spectroscopically silent, which means that we can monitor neither the formation of the phosphorylated product, nor the conversion of ATP to ADP directly using spectroscopy. This is because the products would appear identical to the starting materials due to identical absorption features and extinction coefficients. In order to monitor the activity of your wild type and mutant Abl kinase domains, you will therefore use a coupled assay, which “couples” the conversion of ADP to ATP with the conversion of NADH to NAD+, a process that can be detected by a decrease in absorbance at 340 nm. This coupling assay is outlined in Fig.3. Figure 3. The Kinase Coupled Assay for Abl Activity It is possible to use the kinase coupled assay to generate quantitative kinetic data on the kinase of interest. You will use this assay to determine the specific activity of the wt and mutant Abl kinase domains in the absence and presence of small molecule inhibitors, including Gleevec. Specific activity in biochemistry is defined as the amount of product formed by an enzyme in a given amount of time. Specific activity is often reported in 37units (U) per mg of enzyme, where 1 unit is equal to 1 µmol of product formed per inute. Vis spectrometer. You will record the slope f the resulting line (decrease in absorbance over time) and calculate the specific activity Com in• thawed 1x assay buffer L x µL of Abl kinase domain (typically 2-3 µL for wt Abl- check with your TA) ground read g te indow and the cuvette must be pushed down all the way in the holder. Then add e and the R alue. Subtract your background slope from the slope after substrate addition to ow you can calculate the specific activity of the Abl kinase using this slope determined c ity (th lab or at home) pecifi Activ /mg =mSESSION 13 1.) Determine the specific activity of the wt Abl kinase domain To run the assay, you will set up a 120-µL reaction in a quartz cuvette and monitor the decrease in absorbance at 340 nm on the UV/oof the kinase, as described below. b e the following in a 0.65 eppendorf tube: 86.4 µL of the• 9.6 µL of PK(928 U/mL)/LDH (1300 U/mL) for a final concentration of 74 U/m// 104 U/mL. •• 18-x µL of water Gently mix the solution with tapping and with a pipette, and then transfer the mixed solution to a 100-µL capacity quartz cuvette. Record the initial slope for a backin . Note that for a proper reading the meniscus must not be visible in the cuvetw• 6 uL of 20 mM peptide (for a final concentration of 1 mM) to start the reaction. Let the reaction run for approximately 5 to 10 minutes. Record the slop2vdetermine the slope (change in absorption per minute) from kinase activity. Nin the coupled phosphorylation assay. Calculating the Spe ific Activ is can be calculated in S c ity = U µmol product formed min es: A) the amount of product hosphorylated peptide) formed per minute (in µmol/min) by the Abl kinase and B) the sing Beer’s Law (Abs = εcl) and the slope determined in the assay, calculate the total mg of enzyme added To find specific activity, you need to determine two valu(pamount of Abl enzyme (in mg) that was used in the assay. A) Calculate the amount of product (phosphorylated peptide) formed per minute. U 38concentration of product formed per minute:Beer’s Law can be written: ΔA/min = (Δc/min)(l ε) w ΔA/min = change in absorption per minuthere, e = |slope| ε = the extinction coefficient of your chromophore (here NADH) olving for Δc/min gives the concentration of product formed in units of M/min. Multiply (mol/L). Since the answer should be in terms of µmol/min, OT µM/min, we need to convert µM to µmol. To do this, multiply your answer by the (U). e in the reaction would be 0.00003 mg. If you sed a different stock of enzyme, use the concentration and the volume added to ow you can divide your μM/min of product formed by the total mg of kinase to etermine your specific activity. .) Determine the specific activity of the wt Abl kinase domain in the presence of 1 µM de Gleevec in the reaction mixture: Com in• thawed 1x assay buffer L omain (typically 2-3 µL for wt Abl- check with your TA) 1.2 µL of a 100 µM solution of Gleevec dissolved in DMSO • 16.8-x µL of water l = the path length of the cuvette = 1 cm ε of NADH at 340 nm = 6,220 cm-1M-1 Δc/min = change in concentration per minute. Solve for this value. Sthis value by 106 to get an answer in units of µM/min. Remember that M = molarityNtotal volume of the reaction. Now you have an answer in terms of μM/min, or units B) Calculate the amount of enzyme used in the assay. The Calbiochem wild-type Abl has a concentration of 0.01 mg/mL. If you used 3 µL of this kinase stock, the total amount of enzymudetermine the amount of enzyme (in mg). Nd 2Gleevec. Follow the procedure described in part 1, but inclub e the following in a 0.65 eppendorf tube: 86.4 µL of the• 9.6 µL of PK(928 U/mL)/LDH (1300 U/mL) for a final concentration of 74 U/m// 104 U/mL. • x µL of Abl kinase d• 39Gently mix the solution with tapping and with a pipette, and then transfer the mixed solution to a 100-µL capacity quartz cuvette. Record the initial slope for a background reading. Then add • 6 uL of 20 mM peptide to start the